Composed of polymers of a variety of organic compounds, plastics can be molded, extruded, cast into various shapes and films, and even drawn into fibers. It is such versatility that has led to incorporation of plastics into a seemingly endless number of products. Thus, plastic products have become an integral part of everyday life in industrialized society, and the demand for these products is expected to grow as the world population grows and developing countries move up the economic ladder. However, synthetic plastics are slow to degrade in landfills. If and when they do breakdown, the monomers and their derivatives resulting from degradation may actually be more hazardous to human health than the undegraded polymers (Selenskas et al. (1995) Amer. J. Indust. Med. 28:38R-398; Tosti et al. (1993) Toxicol. Indust. Health 9:493-502; Yin et al. (1996) J. Food Drug Anal. 4:313-318). These concerns have raised to a new level the urgency of exploring the use of the environmentally friendly, compostable polymers as substitutes for synthetic plastics. Polyhydroxyalkanoates (PHAs) are polyesters of hydroxyalkanoic acids that are synthesized by a variety of bacteria as storage polymers under stressful conditions (Steinbuchel, A. (1991) Biomaterials: Novel materials from biological materials, D. Byrom, ed. (New York: Macmillan Publishers Ltd.), pp. 123-213). Since PHAs have thermoplastic properties, that is they become soft when heated and hard when cooled, and are fully biodegradable, they offer an attractive alternative to synthetic plastics (Brandl et al. (1995) Can. J. Microbiol. 41: 143-153; Byrom, D. (1993) Int. Biodeterior. Biodegrad. 31:199-208; Lee, S. Y. (1996) Biotechnol. Bioeng. 49:1-14; Nawrath et al. (1993) Abst. Pap. Amer. Chem. Soc. 206:22-27; Poirier et al. (1995) Bio/technology Nat. Publ. Co. 13:142-150; Steinbuechel, A. (1992) Curr. Opin. Biotechnol. 3:291-297). Unlike man-made plastics, the production of PHA by living organisms is not dependent on finite natural resources like petroleum.
Currently, only one type of polyhydroxyalkanoate (PHA), Biopol, a copolymer made by fermentation, is commercially available (Poirier et al. (1995) Bio/technology Nat. Publ. Co. 13:142-150). However, at approximately $7 per pound, this polymer is much too expensive in comparison to the synthetic plastics that have similar properties but are cheaper with a price of approximately $0.5 per pound (Poirier et al. (1995) Bio/technology Nat. Publ. Co. 13:142-150). The higher cost of Biopol results primarily from its cost of production, the main contributing factor being the substrate (Poirier et al. (1995) Bio/technology Nat. Publ. Co. 13:142-150). If the PHAs can be produced in plants, the cost of production can be lowered substantially because these polymers would compete with seed oil as natural storage constituents of the cell. The current market price of plant seed oil is between 26 and 28 cents per pound (Anonymous (1998) Economic Research Service (Washington, D.C. 20036: U.S. Department of Agriculture). Only about 40% of the energy required to extend a fatty acid chain by two carbons is expended on extending a PHA chain by the same length. Starting with acetyl-CoA, a two carbon extension in oil biosynthesis requires two NADPH and one ATP. In comparison, only one NADPH is needed to accomplish the same for PHA biosynthesis (FIG. 1). Theoretically, more than two units of PHA should be formed for every unit of oil replaced.
Until recently, the only PHA that has been produced in plants was polyhydroxybutyrate (PHB), a homopolymer of 3-hydroxybutyric acid (John et al. (1996) Proc. Natl. Acad Sci. USA 93:12768-12773; Nawrath et al. (1994) Proc. Natl. Acad. Sci. USA 91:12760-12764; Padgette et al. (1997) Plant Physiol. 114 (Suppl.) 3S; Poirier et al. (1992) Science 256.520-523)). Because this polymer is crystalline and brittle with a melting point too close to its degradation point, PHB is difficult to mold into desirable products (Lee, S. Y. (1996) Biotechnol. Bioeng. 491:1-14). Many bacteria make copolymers of 3-hydroxyalkanoic acids with a carbon chain length greater than or equal to five (Steinbuchel, A. (1991) Biomaterials: Novel materials from biological materials, D. Byrom, ed. (New York: Macmillan Publishers Ltd.), pp. 123-213). Such copolymers are polyesters composed of different 3-hydroxyalkanoic acid monomers. Depending on the composition, these copolymers can have properties ranging from firm to elastic (Anderson et al. (1990) Microbiol. Rev. 54:450-472; Lee, S. Y. (1996) Biotechnol. Bioeng 49:1-14). Unlike PHB, the PHA copolymers are suitable for a variety of applications because they exhibit a wide range of physical properties.
Initial attempts at producing PHA in the cytosol proved toxic to the plant (Poirier et al. (1992) Science 256:520-523). This problem was overcome by targeting the PHA-producing enzymes to plastids (Nawrath et al. (1994) Proc. Natl. Acad. Sci. USA 91: 12760-12764). In either cellular compartment, however, only PHB was accumulated, not any of the copolymers. With both of these methods, the genes from Ralstonia eutropha (also known as Alcaligenes eutrophus) were used. The PHA synthase of this bacterium can utilize only short chain (C3-C5) monomers (Steinbuchel, A. (1991) Biomaterials: Novel materials from biological materials, D. Byrom, ed. (New York: Macmillan Publishers Ltd.), pp. 123-213).
Recently, the synthesis of PHA containing 3-hydroxyalkanoic acid monomers ranging from 6 to sixteen carbon in Arabidopsis thaliana was reported (Mittendorf et al. (1998) Proc. Natl. Acad. Sci. USA 95:13397-13402). To accumulate PHA, the Arabidopsis plants were transformed with a nucleotide sequence encoding PHA synthase from Pseudomonas aeuginosa that was modified for peroxisome targeting by the addition of a nucleotide sequence encoding the C-terminal 34 amino acids of a Brassica napus isocitrate lyase. In these plants, PHA was produced in glyoxysomes, leaf-type peroxisomes and vacuoles. However, PHA production was very low in the Arabidopsis plants, suggesting that either the introduced PHA synthase did not function properly in the intended organelle or more likely that the necessary substrates for the introduced PHA synthase were present at levels that were limiting for PHA synthesis. While this report demonstrated that PHA can be produced in peroxisomes of plants, the level of PHA produced in the plants was far below levels necessary for the commercial production of PHA in plants.